1. Field of the Invention
The present invention relates to a process for the determination and detection of an immunologically reactive analyte such as a ligand or ligand receptor in an aqueous sample. More particularly, the invention involves the use of specific binding pairs consisting of immunologically reactive ligands and anti-ligands or ligand receptors which are specifically immunologically reactive, that is bindable, therewith. The invention further relates to kits of materials for use in conducting the process of the invention and to visually determinable or detectable, collected, solid phase, metal-containing composites which are produced in accordance with the procedure.
2. Description of the Prior Art
There has long been a need to measure substances with a high degree of sensitivity and specificity. In particular, in fields such as clinical medicine, forensic science, environmental quality testing, food quality assurance, drug testing and other related areas, the presence and/or amount of trace substances in test samples is often of great significance. In such areas, the measurement of very low concentrations in the order of parts per million or less is sometimes necessary. Moreover, such testing or measurement often requires the identification of particular molecules while not sensing other molecules with similar yet different structures.
The need for sensitive and specific tests has been addressed in the past by the development of a number of immunoassay procedures based on the highly specific and sensitive interaction between an antigen and an antibody directed against such antigen Antigens and antibodies were initially recognized as being the participants in the immune process of an animal, that is, when an animal is injected with a foreign substance that is an antigen (or ANTIbody GENerator), the animal in time responds by producing antibodies which are protein molecules that recognize and tightly bind the invading antigen thereby facilitating removal or destruction of the latter. The immune process is highly specific and the use of immunoassay procedures for identification of specific substances has been exploited with great success. Such procedures have been further facilitated by the important discovery of Milstein and Kohler reported in Nature 256:495-497, 1975, concerning a procedure for preparing so-called monoclonal antibodies The details of this work are well known and there is no need to repeat the same here; however, as a result of the Milstein and Kohler work, the development of highly sensitive and specific reagents has been facilitated.
In the known prior assay procedures referred to as radioimmunoassay (RIA) procedures, either an antibody or an antigen is labelled with a radioisotope such as I.sup.125. In accordance with these known procedures, the amount of the radioisotope in an immune complex may be measured and is a function of the presence or quantity of analyte in the test solution. As is well known in the art, radioimmunoassays may be configured in a variety of ways employing competitive or immune sandwich formats, to name but a few. RIA procedures have been configured for detection of both large analytes such as macromolecules and much smaller substances comprising small molecules such as theophylline. At the present time, any substance which is antigenic or which may be rendered antigenic by coupling to a suitable carrier can be detected by a RIA procedures, and such procedures have been found widespread acceptance, especially in clinical diagnostic laboratories, as a result of the high degree of sensitivity and specificity which can be achieved thereby.
On the other hand, RIA procedures do have some shortcomings which make the use thereof impractical for some types of testing and in some environments. That is to say, RIA procedures require the detection of radioisotopes utilizing sophisticated instrumentation such as gamma or scintillation counters. Moreover, radioisotopes are inherently unstable and have limited shelf life. Additionally, radioisotopes are hazardous and the use thereof is limited to specially trained technicians and laboratories equipped with hazardous waste disposal procedures and facilities.
The shortcomings inherent in RIA procedures have been overcome through the use of non-radioactive labels or markers such as enzyme color formers, fluorescent materials, chemiluminescent markers, etc. When an enzyme is employed as the label, the assay methods have come to be known as enzyme immunoassays (EIA) or enzyme linked immunosorbent assays (ELISA) wherein a solid phase immunosorbent is employed. Commonly, such enzymes as horseradish peroxidase, alkaline phosphatase, glucose oxidase and urease have been employed as labels. These enzymes react with specific substrates to produce a detectable signal, usually production of color, which can be quantitated with somewhat simpler instrumentation, for example colorimeters, than is needed in connection with RIA procedures. Moreover, the use of enzymes presents little or no hazard when compared with radioisotopes.
Most enzyme systems useful in connection with EIA or ELISA procedures are relatively stable and can often be stored under refrigerated conditions for as much as a year or so. As a result, assay procedures utilizing enzyme markers are extensively employed today in a variety of laboratory settings and in some cases in physician's offices and even in the home of the users. Other procedures such as fluorescent and chemiluminescent assays likewise overcome the stability and hazard drawbacks of RIA. However, these procedures generally require sophisticated instruments so that the use thereof is limited primarily to well equipped laboratories. A number of enzyme, fluorescent and chemiluminescent labels useful in immunoassay procedures are disclosed in U.S. Pat. No. 4,233,402.
Generally speaking, enzyme labelled immunoassay procedures have been utilized to satisfy the need and desirability for highly sensitive and specific immunoassays conducted at remote sites. The remote sites where such testing is desirably conducted, as referred to above, include the physician's office and the home of the user. In the physician's office, it is often useful for rapid, simple assays to be performed while the patient is still in the office so that diagnosis may be accomplished without delay and treatment instituted during a single visit. Without such assays, it is often necessary for the physician to collect a sample from the patient during a first visit, such sample then being sent to a clinical laboratory for analysis with the results being reported back to the physician by the laboratory at a later time. In the meanwhile, the patient is sent home and must return for a second visit with the physician in order to receive appropriate treatment and/or medication. Such delay is inefficient and inappropriate and in some cases may even be life threatening.
Home testing is desirable to facilitate testing by the consumer in the privacy of his or her own home. The results of the test may indicate the necessity or lack of necessity of a visit to a physician. Examples of useful tests for the "at home" market are tests for pregnancy, ovulation, streptococcus infection and other infections which may be detected by analysis of urine, saliva or other appropriate test samples.
For remote site testing, assuming appropriate sensitivity and specificity can be achieved, there are at least three other requirements for practical assay procedures. The first of these desirable factors is speed in that the assay must be performed in an acceptably short period of time, the shorter the better. Stability is also a desirable feature in that the components of the assay should be stable for an extended period of time without refrigeration or special handling and the assay results or readout should be sufficiently stable so that the interpretation may be confirmed even several days after the initial test has been performed. Finally, from a commercial view point, it is desirable that the test be as simple as possible requiring only minimal or no instrumentation and precluding mistakes and poor performance resulting in incorrect interpretations.
Immunoassay kits employing enzyme markers are available commercially today for determining pregnancy and ovulation. The technical components generally included in such kits are (1) a solid phase bearing immobilized antibody, (2) an enzyme labelled antibody, (3) a rinse solution (in some cases this may be the user's tap water), and (4) a substrate for the enzyme. A typical procedure is that the sample is mixed with the solid phase and incubated (with or without a subsequent rinse step) and then the sample is discarded. The solid phase is then contacted with the enzyme labelled antibody and incubated and thereafter the solid phase is rinsed and contacted by the substrate. After a period of time (ca. 5 minutes) the color of the solid phase is observed One such assay is described in U.S. Pat. No. 4,632,901.
Enzyme labelled immunoassays are not without their own drawbacks resulting from the instability of sole enzyme systems, the number of kit components and the complexity of the procedure. As a result, work continues in an effort to simplify, increase the speed of and provide stability for the components and products of immunoassay procedures, particularly procedures to be conducted at remote sites. One result of such work was the recognition of metal sol particles as a marker in an immunoassay procedure. Such procedure is disclosed in U.S. Pat. No. 4,313,734. In this patent disclosure, protein coated metal sol particles react with a protein coated solid phase to cause a change in optical properties and provide a colorimetric determination in the liquid phase. The immunological reaction results in an agglutination or agglomeration of dispersed materials, an occurrence which brings about changes in the light absorption and reflection characteristics of the liquid phase. Such changes are measured using instruments such as spectrophotometers. In some cases, the color change in the liquid phase is such that it may even be assessed visually by comparison of the coloration in the sample with the coloration of control liquids, and even perhaps by observing subtle color changes in the fluid as the coloration changes from red to purple to colorless. The course of the reaction in an agglutination test is time dependent and the visual determinations must be made at a particular point in time. Moreover, the test results generally lack stability since the reaction continues even after visual assessment is made.
Agglutination resulting from the reaction of immunoreactive reagents and utilizing gold as a label was also used for the detection of mannan by Horisberger and Rosset, in their work described in their article entitled "Colloidal Gold, A Useful Marker For Transmission And Scanning Electron Microscopy", J. Histochem. Cytochem. 25, 1977, pp. 295-305. In this work, an agglutination process was employed and the course of the immunoreaction was followed by spectrophotometrically reading the absorbance of light in the fluid. This work provided a prelude for the work described in the '734 patent.
Another prior procedure which utilized protein coated particles is described in U.S. Pat. No. 4,115,535. The procedure described in the '535 patent involves an immunological process which results in agglutination of two different kinds of particles coated with the same protein. One of the particles has magnetic properties so that the agglutinated mass may be separated from the liquid with a magnet. The other kind of particle is described as consisting of fluorescent and/or distinctively colored small polymer particles. The size of each of the particles is specified as being one micron or less. Although metal particles are used as magnetic particles, there is no suggestion in this disclosure of the use of metal particles to provide a colorimetric result. Agglutination is also involved in the procedure disclosed in U.S. Pat. No. 4,486,530.
An interesting prior art procedure is disclosed in the published Abstracts of the Annual Meeting - 1986 of the International Congress of Immunology, at page 363 (Abstract C-213). This procedure involves the use of antibodies immobilized on a nitrocellulose membrane. The antibodies are used to capture antigens during immunofiltration of the specimen through the membrane. Thereafter the membrane was stained with colloidal gold conjugated antibodies. A red spot was said to indicate a positive reaction. This procedure suffers from the same sort of defect as the enzyme immunoassay procedures described above in that two pouring steps are required and the procedure requires the prior preparation and use of a membrane to which specific antibodies are bound.